Molecular Stringency in Multiplexed Assays—
A self-complementary oligonucleotide capture probe in a “looped” configuration may be used to adjust molecular stringency in an assay. Assay stringency relates to the positive results produced by an assay, such that high stringency conditions generate relatively fewer positive results than lower stringency conditions. Looped probes are described in WO 01/98765, entitled: “Multianalyte Molecular Analysis Using Application-Specific Random Particle Arrays” and U.S. Pat. No. 6,361,945 (assigned to Gen Probe, Inc.). Such a probe consists of a 5′-terminal subsequence and a complementary 3′-terminal subsequence, tethered by an unrelated subsequence, the two terminal subsequences capable of forming a duplex (“stem”), and the tether forming a loop, and either the 5′-terminal subsequence of the 3′-terminal subsequence capable of forming a duplex with a target nucleic acid. The probe may be attached to a solid phase such as an encoded microparticle (“bead”), by way of an appropriate functional modification of the 5′terminal subsequence or the loop subsequence.
Using a fluorescence acceptor and a proximal fluorescence quencher (as discussed in U.S. Pat. No. 6,534,274), capture of a target nucleic acid is detected by way of detecting a transition from the Closed (“C”) state of the capture probe to the Open (“O”) state or the target-associated (“OT”) state, the O-state contributing to “background” fluorescence, independent of target concentration (FIG. 1). In this competitive equilibrium, low stringency, favoring the closed state, will reduce the likelihood of formation of the open (or other intermediate state, see Detailed Description, below) required for probe-target duplex formation, thereby diminishing the detection sensitivity. Conversely, high stringency, favoring the open state, will likewise reduce the likelihood of target capture—by reducing the stability of any probe-target duplex—while producing indiscriminate fluorescence, independent of captured target, thereby reducing specificity.
Thus, the use of a looped probe calls for resolution of the conflict between detection sensitivity and specificity, preferably by operating near an optimal stringency, determined by a choice of buffer conditions and operating temperature. For typical buffer conditions, which generally are of low ionic strength, e.g. corresponding to salt concentrations of 50 mM, this step requires selection of an optimal detection temperature, preferably at or above the range of the midpoint of the melting curve where specificity is optimal. Optimal stringencies generally will depend on capture probe sequences, and on target configuration and/or length. Thus, identifying the optimal stringency range in a multiplexed assay thus becomes increasingly difficult with each different probe added, given the dispersion of the melting curve profiles of a set of different probe-target complexes under given assay conditions.